Figure 5.

PGT regulates EP₄ receptor desensitization in response to exogenous PGE₂. Wild-type MDCK (“WT”) and PGT-GFP-MDCK (“PGT-MDCK”) cells were transiently transfected with the EP₄-expressing cDNA, and 24 h later were exposed to pharmacological concentrations of exogenous PGE₂ (0–50 nmol/L, “applied medium [PGE₂]”) in the presence or absence of 5 μmol/L T26A. After 10 min, media were collected for determining residual extracellular PGE₂ concentrations by ELISA, and cells were harvested for quantifying the amount of cell surface EP₄ receptor using a binding assay. (A) Cell surface EP₄ receptor. Exogenous PGE₂ caused a reduction in cell surface EP₄ (i.e., desensitization) in wild-type MDCK cells (open circles); T26A had no effect on this desensitization (closed circles). When PGT was expressed, it abrogated the ability of PGE₂ to cause EP₄ desensitization (open triangles), an effect that was reversed by the PGT inhibitor T26A (closed triangles). (B) Media [PGE₂] at the conclusion of 10 min exposure to cell monolayers. PGT reduced the concentration of PGE₂ in the media (open triangles) compared to the lack of change in wild-type cells (open circles) or wild- type cells exposed to T26A (closed circles). T26A reversed the ability of PGT to lower media [PGE₂] (closed triangles). (C) Intracellular cAMP accumulation. PGT-GFP-MDCK cells (“PGT-MDCK”) were transfected with EP₄ as above, treated with 250 μmol/L IBMX for 18 h, and exposed to exogenous PGE₂ ± T26A for 10 min at various doses as above. Cells expressing uninhibited PGT (absent T26A, open triangles) exhibited a robust cAMP dose-response to PGE₂, whereas PGT-MDCK cells in which PGT was inhibited by T26A (open triangles) exhibited a significantly blunted cAMP response to exogenous PGE₂. Values are mean ± SEM (n = 3 for each experiment). *P < 0.05.